| Clostridium perfringens(C.perfringens) is a Gram-positive bacillus commonly found in gastrointestinal tract of humans and animals or soil will cause variety disease in both human and animals including gas gangrene, food poisoning, necrotizing enterocolitis and enteritis necroticans. C.perfringens can be classified to five biotypes (type A to E) based on the exotoxin it secreted. Gas gangrene which mainly caused by C.perfringens type A infection is a rapid and the most destructive disease. Once infected over certain amount of C.perfringens type A, it will rapidly extend to the whole body even harm to the life. During World War II, people infected by C.perfringens because of combat wound made gas gangrene spread as plague. At present, gas gangrene usually occurre in earthquakes disaster or traffic accidents. In clinic, because of the uncontrolled characteristic of gas gangrene, patients usually were amputated to hold the life but severely be hurt and loss labor capacity.Due to the rapid destruction of gas gangrene, a quick and sensitivety detection method which help to early diagnosis shall be established.The previously described methods for detecting C.perfringens including cultivation, biochemistry identifica-tion system, enzyme linked immunoassays. plasmid analyses ect. These techniques have been used in dection and identification schemes followed by isolation or cultivation, those will largely increase the time to obtain positive results. Consider-ation of the problems present existing, we aim to study the application value of C.perfringens real-time PCR method by establishment of mouse gas gangrene model.Part I:C.perfringens real-time PCR method16SrDNA gene has bacame the most stable molecular marker in bacetrium detection as its high converstion in species. To establish the C.perfringens real-time PCR method, we designed the primers and probe based on the conservative region of16SrDNA and successful amplicated the target fragement with the length of105bp BLAST analysis show that the sequencing result is identical to the gene of C.perfringens16SrRNA [AB566417]. A specical probe were designed by the target fragment and the real-time PCR system was optimized to test the sensitivity and stability by detecting C.perfringens with14different concentration ranged from1014cfu/mL to101cfu/mL. As a result, the concentration of C.perfringens ranged from1014cfu/mL to101cfu/mL can be successful detected by real-time PCR with the low detection limit of101cfu/mL. The mean CV of Ct values with parallel test is0.014±0.014, and95%CI is (0.006-0.022).Part Ⅱ:Virulence test of ATCC13124C.perfringens Type A is the main biotype leads to gas gangrene. ATCC13124, one of representative strain of C.perfringens, was orginally isolated from a gas gangrene patient can produces large quantity of gas gangrene-associated toxin. Before established the model, the virulence of the ATCC13124should be measured by animal expriment. The virulence tests were including exotoxin virulence test and bacterial virulence test.In the exotoxin virulence test, we obtain the bacterium exotoxin in the superna-tant afer centrifugation from culturing in PY medium. Mice were grouped to received different volume of exotoxin related to weight by tail intravenous injection and the LD50is13.3mL/kg and95%CI is (11.4～15.7) mL/kg calculated by Bliss method.In the bateria virulaence test, mice were grouped to receive different concentration of ATCC13124(5.7×109cfu/mL,5.7×108cfu/mL;5.7×07cfu/mL,5.7×106cfu/mL and5.7×105cfu/mL) with1mL by abdominal injection and the dead rate, distribution of survival time and the infectious status were observated. The dead rate of5.7×109cfu/mL groupn5.7×108cfu/mL group、5.7×107cfu/mL group、5.7×106cfu/mL group、5.7×105cfu/mL group were100%,90%,10%0%,0%and0%in order and the whole dead rate have no significant difference (χ=51.900, P<0.001).The dead rate of control group has significant difference compared with both5.7×109cfu/mL group (χ2=20.000, P<0.001, corrected P<0.001) and the5.7×108cfu/mL group (χ2=16.364, P<0.001, corrected P<0.001), but has no significant difference compared with5.7×107cfu/mL group (χ2=1.053. P=0.305, corrected P=1.000).5.7×109cfu/mL group has no significant difference compared with5.7×108cfu/mL group (χ2=1.053. P=0.305, corrected P=1.000), but has significant difference compared with5.7×10cfu/mL group (χ2=16.364, P<0.001, corrected P<0.001),5.7×106cfu/mL group (χ2=20.000, P<0.001, corrected P<0.001),5.7×105cfu/mL group (χ2=20.000, P<0.001, corrected P<0.001).The dead rate of5.7×108cfu/mL group has significant difference compared with5.7×107cfu/mL group (χ2=12.800, P<0.001, orrected P<0.001),5.7×106cfu/mL group (χ2=16.364, P<0.001, corrected P<0.001),5.7×105cfu/mL group (χ2=20.000, P<0.001, corrected P<0.001). The dead rate of5.7x10’cfu/mL group has no significant difference compared with5.7×106cfu/mL group (χ2=1.053, P=0.305, corrected P=1.000),5.7×105cfu/mL group (χ2=1.053, P=0.305, corrected P=1.000).The median survival time of5.7×109cfu/mL group is1.000h with95%CI is (1.000～2.167) h. The median survival time of5.7×108cfu/mL group is2.333h with95%CI is (1.688～2.797) h. The survival time has significant diffenence among the whole test group by Log rank method (χ2=32.963, P<0.001). Pairwise comparision shows that there are significant difference in5.7×10cfu/mL group and5.7×108cfu/mL group (χ2=9.886, P=0.002),5.7×109cfu/mL group and5.7×107cfu/mL group (χ2=22.550, P<0.001),5.7×108cfu/mL group and5.7×107cfu/mL group (χ2=14.875, P<0.001).The appearance in skin of mice abdominal inject with5.7×109cfu/mL and5.7×108cfu/mL changes from pale to bronze and then to purplish red, whole body toxemia, expanded air-filled in abdomen smell with hydrogen sulfide after anatomy, and intestinal tract with a large of gas. The mice abdominal injected with5.7×107cfu/mL show slightly pale in skin and filled with a little gas in intestinal tract. Mice still alive in this group show no infection.Part Ⅲ:Establishment and application of mice gas gangrene model by C.perfringens real-time PCRWe choice the ATCC13124which is the representative strain of C.perfringens and kunming mouse of SPF level which can be statistical analyzed to establish a gas gangrene model in order to support an animal model to study the application value of C.perfringens real-time PCR.We utilize intramuscular injection method to establish a gas gengrene model at single infection and atraumatic state. First, we prepare a series concentration of ATCC13124range of3.5×109cfu/mL、3.5×108cfu/mL、3.5×107cfu/mL, then intramuscular inject in the mouse leg with0.1mL and the control group with0.1mL sodium chloride then observation the process of bacterium infection.The dead rate of5.7×109cfu/mL group、5.7×108cfu/mL group、5.7×107cfu/mL group and the control group are100%,70%,10%and0%in order and the whole dead rate have no significant difference (χ2=27.879, P<0.001).The dead rate of control group has significant difference compared with3.5×109cfu/mL group (χ2=20.000, P<0.001, corrected P<0.001) and3.5×108cfu/mL group (χ2=10.769, P=0.001, corrected P=0.006) but has no significant difference compared with the3.5×107cfu/mL group (χ2=1.053,P=0.305, corrected P=1.000).The dead rate of3.5×109cfu/mL group has no significant difference compared with the3.5×108cfu/mL group (χ2=3.529, P=0.060; corrected P=0.360) but has significant difference compared with the3.5×107cfu/mL group (χ2=16.364, P<0.001. corrected P<0.001).The dead rate of3.5×108cfu/mL group has significant difference compared with the3.5×107cfu/mL group (χ2=7.5; P=0.006, corrected P=0.036).The median survival time of5.7×109cfu/mL group is15.000h with95%CI is (11.901～18.099) h. The median survival time of5.7×108cfu/mL group is21.000h with95%CI is (14.802～27.198) h.The survival time has significant diffenence among the whole test group by Log rank method (χ2=20.922, P<0.001). Pairwise comparision show that there has significant difference in5.7×109cfu/mL group and5.7×108cfu/mL group (χ2=4.062, P=0.044),5.7×109cfu/mL group and5.7×107cfu/mL group (χ2=20.284, P<0.001),5.7×108cfu/mL group and5.7×107cfu/mL group (χ2=8.006, P<0.005).Different concentration of ATCC13124can lead to different sysptom. Mice intramuscular inject with3.5×109cfu/mL appear flare and rigor and can not move in the beginning12hour and then the flare was extenting from the lower limbs to the upper limb and the cohort with purple red and congestion in the skin with toxemia in the whole body. The infection part filled with large amount of gas and smell as hydrogen sulfide after dissected. All the dead mice appeared hematuria. The syptom of dead mice received3.5×108cfu/mL is similar to the3.5×109group but lighter appeared flare and rigor without movement and then the flare was extenting from the lower limbs to the upper limb and the cohort with purple red and congestion in the skin without toxemia in the whole body. The infection part filled without gas and none of dead mice appeared hematuria. The syptom of alive mice appeared flare but recovered during48h. The group received3.5×10’cfu/mL only one mouse died and the syptom as3.5×108cfu/mL group. The survival mice received3.5x10’cfu/mL has appeared flare at the beginning but recovered during36h. The control group show normal without any syptom.To verify the animal model wether it is infected by C.perfringens, we use traditional method such as gram staining, culture and the anaerobe identify system. The secretions were obtained from the part of mice intramusular injection with different concentration of ATCC13124. Both3.5×109group,3.5×108group and the dead mouse in3.5×10’ group can find the gram-positive rod bacterium with spore forming in the one side, can culture anaerobe with a clear zone of hemolysis. Neither survival mice in3.5×10’ group nor the control group can find the gram-positive bacterium or culture any anaerobe. Anaerobe after cultured were identified to C.perfringens by API20A identify system.The secretion of gas gangrene models were detected by C.perfringens real-time PCR. All the expremental groups were decteded as positive results while the control group was negative. The mean Ct value of3.5×10cfu/mL group,3.5×108cfu/mL group and the3.5×10’cfu/mL group were21.208±2.693,28.454±2.739and 32.489±2.874in order with significant difference (F=42.592, P<0.001). Multiple comparison show that there are significant difference in3.5×109cfu/mL group and3.5×108cfu/mL group (P<0.001),3.5×109cfu/mL group and3.5×107cfu/mL group (P<0.001),3.5×108cfu/mL group and3.5×107/mL group (P=0.003).ConclusionsC.perfringens real-time PCR which primer and probe were designed accroding to16SrDNA can detecte the concentration range from1014cfu/mL to101cfu/mL with good stability in the reaction system and the low detection limit is101cfu/mL.We have a primary study on the exotoxin of ATCC13124. Utilizing PY medium can recover the virulence of ATCC13124from dormancy.Mice abdominal injected with ATCC13124at the cocentretion of109cfu/mL and108cfu/mL can lead to rapid dead. The cocentretion of ATCC13124higher than108cfu/mL can lead mice to entro-gangrene when higher than109cfu/mL can lead mice100%mortality, while lower than107cfu/mL hardly infected.Intramuscular injected with ATCC13124at the concentration of108cfu/mL and109cfu/mL for0.1mL can establish the mouse gas gangrene model. Intramuscular injected with ATCC13124at the concentration of108cfu/mL can lead to70%mice suffer from gas gangrene and died during27h while intramuscular injected with109cfu/mL for0.1mL can lead to100%mice suffer from gas gangrene and died during18h, but lower than10’cfu/mL hardly infected.C.perfringens real-time PCR method is superior to the gram-staining and culture method. Ct value can infer the concentration of C.perfringens in gas gangrene in order to guide clinical diagnosis. When the test result is positive but with high Ct value infered that infection of C.perfringens with a low concentration or has been died, and mice can remove the bacterium by immune system will not lead to gas gangrene. So doctor should diagnose with clincal symptom when obtain a test result of a high Ct value by C.perfringens real-time PCR. |
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